An Immunological Role in the CARDs

Courtesy of Gabriel Nuñez AND NOW PRESENTING: In this model, microorganisms phagocytosed by the antigen presenting cell (left) release ligands recognized by NOD/CARD proteins and peptides that associate with MHC class II molecules. Recognition of intracellular ligands by NOD/CARD proteins and extracellular ligands by Toll-Like Receptors (TLRs) mediates NF-kB activity. These intracellular events induce cytokine secretion and antigen presentation, as well as the expression of co-stimulatory molecules. It's rare in this feature to highlight a researcher's work twice in the same year, because it is rare for a researcher to achieve what Gabriel Nuñez's team at the University of Michigan, Ann Arbor, has accomplished. Besides generating at least three Hot Papers on NOD1 and NOD2 in a matter of a few months,1-3 Nuñez helped create the discipline of intracellular pathogen recognition, and influenced the work of scientists in fields as diverse as genetics, innate and mucosal immunity, gastroenterology, and apoptosis research. Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. N. Inohara et al., "Human NOD1 confers responsiveness to bacterial lipopolysaccharides," J Biol Chem, 276:2551-4, 2001. (Cited in 128 papers) Y. Ogura et al., "NOD2, a NOD1/Apaf-1 family member that is restricted to monocytes and activates NF-kB," J Biol Chem, 276:4812-8, 2001. (Cited in 153 papers) Prior to publishing Hot Papers that connected NOD2 mutations and Crohn disease,1 Nuñez and others demonstrated that NOD1 and NOD2 (now officially known as CARD4 and CARD15, respectively) play a role in innate immunity. In these two papers,2,3 Nuñez's team published results arguing that CARD4 and CARD15, both presumably cytosolic proteins, recognize the bacterial cell wall constituent lipopolysaccharide (LPS).2 Further, activating either protein stimulates the immunoregulatory transcription factor NF-kB.3 LPS recognition was initially criticized. And these results ultimately turned out to be based on contaminated reagents, proving the critics correct. Yet, it spurred further work and kept this group, as one researcher put it, "barking up the right tree." John Reed, researcher and president of the Burnham Institute, La Jolla, Calif., says these proteins could have a future. "We don't know nearly as much as we need to about the biochemistry of how they really function. As the dust settles and the data come in, there'll be some real targets in here for drug discovery that will be quite exciting." AN INTRACELLULAR TLR? CARD4 was supposed to be a regulator of apoptosis, period. Nuñez's lab found it in 1999 by searching for structural homologs of the apoptotic protease activating factor-1 protein (Apaf-1). They published evidence that it binds many caspase proteases and activates at least one.4 Apaf-1 and CARD4 share a tripartite domain structure. An N-terminal effector-binding domain may help recruit proteins such as proteases or kinases. In the center, a nucleotide-binding oligomerization domain (NOD) is thought to promote the protein's self-oligomerization, which is needed to activate downstream effector molecules. Apaf-1 and CARD4 have a caspase-recruitment domain (CARD) at their C-terminals, but Apaf-1's is made up of WD40 repeats, while CARD4's is of leucine-rich repeats (LRRs). They both belong to a large family of proteins with a similar tripartite structure. But, the intracellular CARD4 could activate NF-kB. This function, perhaps more so than its apoptotic role, caught the attention of Nuñez as well as John Bertin's group at Millennium Pharmaceuticals, Cambridge, Mass. They simultaneously cloned the molecule.5 Says Nuñez: "NF-kB is a transcription factor involved in the inflammatory response ... [with] many functions, including regulation of inflammation in response to pathogens." The Toll-like receptors (TLRs), which are the mammalian cell-surface pathogen sensors, induce proinflammatory cytokines by activating intermediaries such as NF-kB. But researchers found no such receptors in the mammalian cytosol. Philippe Sansonetti and Dana Philpott's studies on Shigella flexneri at the Institut Pasteur had suggested that bacteria could activate epithelial cells by a TLR-independent pathway. "I was really looking for an intracellular Toll-like receptor, basically; something that could be cytoplasmic and could also be implicated in the activation of NF-kB," Philpott explains. Nuñez's and Bertin's papers4,5 looked like the answer, she says. ACCEPTANCE NOT IN THE CARDS It should have come as no surprise when Nuñez, in 2000, found that CARD4 and CARD15 conferred responsiveness to LPS. But peers received the results with skepticism, he recalls. "We had some problems publishing the paper because of that." Indeed, Bruce Beutler of the Scripps Research Institute, La Jolla, Calif., panned Nuñez's LPS-recognition claims. Beutler, whose work helped to establish TLR4 as the extracellular LPS receptor, wrote, "So far, there is no 'strong' evidence to support a direct role for [CARD4] or [CARD15] in LPS sensing ... [this] would entail: (1) a well controlled demonstration that chemically synthesized LPS is sensed when injected into the cytoplasm of cells from mice lacking TLR4, and (2) demonstration that mice lacking [CARD] proteins cannot respond to the same stimulus."6 He suggested that perhaps these molecules could be acting downstream of the actual receptor, but no data yet provide compelling evidence. Despite the initial reticence, the resulting paper, the first of this issue's Hot Papers, was highly cited in the ensuing two years.2 It showed that cells transfected with CARD4 or CARD15 could upregulate NF-kB activation in response to bacterial lipopolysaccharides, indicating that an intracellular mechanism exists for bacterial recognition. The second of this issue's Hot Papers, which was submitted before, but published after the first, describes CARD15. With two CARD domains, it is restricted to the monocyte lineage.3 The paper shows that CARD15's CARD domains interact with the CARD domain of the serine-threonine kinase RICK, leading ultimately to NF-kB activation. The same interactions had previously been shown for CARD4,4,5 implying that the two CARDs may affect the same downstream pathways. DISEASES LINKED Later in 2001, groups showed that mutations in CARD15 conferred susceptibility to Crohn disease, a chronic inflammatory bowel disorder.1 This discovery lent new fodder to those who believed that the malady had a microbial (as opposed to a strictly autoimmune) etiology. Geneticist Jean-Pierre Hugot at the Fondation Jean Dausset in Paris, whose lab also discovered the link between CARD15 and Crohn, says he believes researchers now generally accept that bacteria are malefactors of Crohn disease. Beutler, while acknowledging the connection to bacteria, wrote, "There is no concrete proof of a microbial 'first cause.'"6 Soon after, Hugot's group linked mutations in CARD15 to Blau syndrome, a monogenic inflammatory disorder. Since then, related proteins have been associated with cold autoinflammatory syndrome, Muckle-Wells syndrome (cryopyrin), and familial Mediterranean fever (pyrin). Reed calls them "a very exciting family of proteins," which likely work in roughly the same way, except that the LRRs probably respond to different molecular determinants. They are "very important components of innate immunity," which, Reed speculates, "will likely be involved in a variety of inflammatory diseases ... I suspect it's just the tip of the iceberg." PROVING THEIR CRITICS RIGHT Differing molecular determinants for the NODs and their growing family would give a different repertoire of sensors within the cell, allowing the host to appropriately respond to numerous pathogens. But, notes Reed, it has not been formally demonstrated that any other family members can recognize microbial products. Attempts to identify microbial products recognized by CARD4 and CARD15 themselves have had a rocky history. "We use the word 'recognize' loosely," points out microbiologist Daniel Portnoy, University of California, Berkeley. "No one has shown yet actual biochemical recognition." It turns out that Beutler and others who questioned the NOD's ability to recognize LPS were right. Work earlier this year by Nuñez, Philpott, and others has shown that CARD4 and CARD15 were actually responding to small fragments of peptidoglycans that had contaminated the LPS preparation used as the basis of the original reports.7-10 CARD4 responds to a dipeptide (iE-DAP) found primarily in Gram-negative, and in some Gram-positive, bacteria. CARD15 now has been shown to react to muramyl dipeptide, a conserved structure found in the cell wall of virtually all bacteria. To which component the CARD4 or CARD15 responds was a "small detail," Nuñez says. "The main finding was that these molecules are used for recognition of bacteria." But Portnoy, who was among those who couldn't reproduce the LPS results, maintains this was more than minutiae. Portnoy's complaint: The Hot Papers, he says, were significant but not particularly strong. "The subsequent papers I think were stronger ... so they are barking up the right tree there." Josh P. Roberts (tcwriter@msn.com) is a freelance writer in Minneapolis. References 1. L. Pray, "Researchers put linkage disequilibrium on the map," The Scientist, 17[3]:30-1, 2003. 2. N. Inohara et al., "Human NOD1 confers responsiveness to bacterial lipopolysaccharides," J Biol Chem, 276:2551-4, 2001. (Cited in 128 papers) 3. Y. Ogura et al., "NOD2, a NOD1/Apaf-1 family member that is restricted to monocytes and activates NF-kB," J Biol Chem, 276:4812-8, 2001. (Cited in 153 papers) 4. N. Inohara et al., "NOD1, an Apaf-1-like activator of caspase-9 and nuclear factor kB," J Biol Chem, 274:14560-7, 1999. 5. J. Bertin et al., "Human CARD4 protein is a novel CED-4/Apaf-1 cell death family member that activates NF-kB," J Biol Chem, 274:12955-8, 1999. 6. B. Beutler, "Autoimmunity and apoptosis: the Crohn's connection," Immunity, 15:5-14, 2001. 7. M. Chamaillard et al., "An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid," Nat Immunol, 4:702-7, 2003. 8. S.E. Girardin et al., "NOD1 detects a unique muro- peptide from Gram-negative bacterial peptidoglycan," Science, 300:1584-7, 2003. 9. N. Inohara et al., "Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease," J Biol Chem, 278:5509-12, 2003. 10. S.E. Girardin et al., "Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection," J Biol Chem, 278:8869-72, 2003. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

An Immunological Role in the CARDs

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